Prostate cancer is the second leading cause of death in men in the US. Testosterone (T) - or dihydrotestosterone (DHT) -activated androgen receptor (AR) plays a critical role in prostate cancer during all stages. Castration removes circulating T t deprive prostate cancer cells of T and DHT, and is the preferred treatment for symptomatic high-risk locally advanced or metastatic prostate cancer. Castration-treated prostate cancer initially responds well, but inevitably progresses to castration-recurrent prostate cancer (CRPC), which is incurable and usually fatal. AR signaling remains a predominant driving force for disease progression even after castration. CRPC are able to produce T or DHT through intracrine androgen metabolism that uses cholesterol or circulating adrenal androgens dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Castration does not significantly reduce circulating DHEA and DHEAS. Serum concentrations of DHEAS are >400-fold higher than those of DHEA. DHEAS is much closer to the final products than cholesterol to provide a more energy-efficient substrate for synthesis of T or DHT. Also, DHEAS has a longer half-life in the serum than DHEA. Therefore, DHEAS presents a potential source of precursor that is highly abundant for intracrine production of T or DHT by CRPC cells. However, DHEAS has received little attention. The question of how DHEAS is made available to and used by prostate cancer cells is critically important. The uptake of DHEAS by prostate cancer cells may be mediated by a special class of transmembrane transporters. Steroid sulfatase (STS) is required to hydrolyze DHEAS to DHEA for T and DHT production, and may be regulated by insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R). The central hypothesis of the proposed research is that depriving prostate cancer cells of DHEAS will improve response to castration and prevent the progression to CRPC. The central hypothesis will be tested in 3 specific aims. Aim 1 addresses clinical relevance of STS, IGF1R, and IR in CRPC using immunohistochemistry on sections of a set of tissue microarrays. Aim 2 evaluates the value of targeting DHEAS usage by prostate cancer cells to prevent post-castration tumor growth using in vivo models and inhibition of STS and IGF1R/IR. Aim 3 identifies the most potent DHEAS uptake transporters and tests the feasibility of using a cell model to screen for DHEAS transporter blockers. The proposed studies are required to validate the concept that DHEAS is an important source of precursors for intracrine production of T and DHT by prostate cancer cells. Findings from the studies will provide insight into how to block the use of DHEAS by prostate cancer cells to facilitate more complete androgen deprivation therapy by targeting DHEAS transporters, STS, and STS regulators.